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Abstract

Fluorescence correlation spectroscopy (FCS) is used to study the movements and the interactions of
biomolecules at extremely dilute concentrations, yielding results with good spatial and temporal
resolutions. Using a number of technical developments, FCS has become a versatile technique that
can be used to study a variety of sample types and can be advantageously combined with other methods.
Unlike other
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uorescence-based techniques, the analysis of FCS data is not based on the average
intensity of the
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uorescence emission but examines the minute intensity
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uctuations caused by sponta-
neous deviations from the mean at thermal equilibrium. These
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uctuations can result from variations
in local concentrations owing to molecular mobility or from characteristic intermolecular or intramo-
lecular reactions of
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uorescently labeled biomolecules present at low concentrations. Here, we provide
a basic introduction to FCS, including its technical development and theoretical basis, experimental
setup of an FCS system, adjustment of a setup, data acquisition, and analysis of FCS measurements.
Finally, the application of FCS to the study of lipid bilayer membranes and to living cells is discussed.